Quantitative Proteomic Analysis Reveals Changes in the Benchmark Corynebacterium pseudotuberculosis Biovar Equi Exoproteome after Passage in a Murine Host

Corynebacterium pseudotuberculosis biovar equi is the etiologic agent of ulcerative lymphangitis. To investigate proteins that could be related to the virulence of this pathogen, we combined an experimental passage process using a murine model and high-throughput proteomics with a mass spectrometry, data-independent acquisition (LC-MSE) approach to identify and quantify the proteins released into the supernatants of strain 258_equi. To our knowledge, this approach allowed characterization of the exoproteome of a C. pseudotuberculosis equi strain for the first time. Interestingly, the recovery of this strain from infected mouse spleens induced a change in its virulence potential, and it became more virulent in a second infection challenge. Proteomic screening performed from culture supernatant of the control and recovered conditions revealed 104 proteins that were differentially expressed between the two conditions. In this context, proteomic analysis of the recovered condition detected the induction of proteins involved in bacterial pathogenesis, mainly related to iron uptake. In addition, KEGG enrichment analysis showed that ABC transporters, bacterial secretion systems and protein export pathways were significantly altered in the recovered condition. These findings show that secretion and secreted proteins are key elements in the virulence and adaptation of C. pseudotuberculosis. Collectively, bacterial pathogenesis-related proteins were identified that contribute to the processes of adherence, intracellular growth and evasion of the immune system. Moreover, this study enhances our understanding of the factors that may influence the pathogenesis of C. pseudotuberculosis.Fil: Marques Da Silva, Wanderson. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; Brasil. Institut National de la Recherche Agronomique; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Carvalho, Rodrigo D. De Oliveira. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; BrasilFil: Dorella, Fernanda A.. Universidade Federal de Minas Gerais; BrasilFil: Folador, Edson L.. Universidade Federal da Paraíba. Centro de Biotecnologia; BrasilFil: Souza, Gustavo H. M. F.. Waters Corporation; BrasilFil: Pimenta, Adriano M. C.. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; BrasilFil: Figueiredo, Henrique C. P.. Universidade Federal de Minas Gerais; BrasilFil: Le Loir, Yves. Institut National de la Recherche Agronomique; FranciaFil: Silva, Artur. Universidade Federal do Pará; BrasilFil: Azevedo, Vasco. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; Brasi

A combined approach for comparative exoproteome analysis of Corynebacterium pseudotuberculosis

Background: Bacterial exported proteins represent key components of the host-pathogen interplay. Hence, we sought to implement a combined approach for characterizing the entire exoproteome of the pathogenic bacterium Corynebacterium pseudotuberculosis, the etiological agent of caseous lymphadenitis (CLA) in sheep and goats. Results: An optimized protocol of three-phase partitioning (TPP) was used to obtain the C. pseudotuberculosis exoproteins, and a newly introduced method of data-independent MS acquisition (LC-MSE) was employed for protein identification and label-free quantification. Additionally, the recently developed tool SurfG+ was used for in silico prediction of sub-cellular localization of the identified proteins. In total, 93 different extracellular proteins of C. pseudotuberculosis were identified with high confidence by this strategy; 44 proteins were commonly identified in two different strains, isolated from distinct hosts, then composing a core C. pseudotuberculosis exoproteome. Analysis with the SurfG+ tool showed that more than 75% (70/93) of the identified proteins could be predicted as containing signals for active exportation. Moreover, evidence could be found for probable non-classical export of most of the remaining proteins. Conclusions: Comparative analyses of the exoproteomes of two C. pseudotuberculosis strains, in addition to comparison with other experimentally determined corynebacterial exoproteomes, were helpful to gain novel insights into the contribution of the exported proteins in the virulence of this bacterium. The results presented here compose the most comprehensive coverage of the exoproteome of a corynebacterial species so far

POSSÍVEIS REPRESENTAÇÕES SOBRE CIÊNCIA NA LEITURA DE RECORTES DE TEXTOS DE CIENTISTAS E FILÓSOFOS

It is common in our society, even in school situations, to speak of science as if it was unique. Results of scientific production are often valued without addressing the way they are produced. A possible imaginary about the existence of a unique scientific method may derive from it. This article starts from the realization of the relevance of questioning positions that can value science and the scientific method as if they were unique, that is, positions that do not consider at the multidisciplinary character of this institution. We seek to understand some representations of scientists and philosophers on this issue. These representations are inferred in the reading of discourses present in clippings of their productions, and these clippings are selected in view of possible reflections on the questioning pointed out. For the development of reading as well as for the notion of representation we based on principles and notions of discourse analysis in the Pecheutian strand, mainly in Eni Orlandi’s productions. In this strand discourse is considered as the effect of meanings between interlocutors. Among the results of discourse analysis present in these clippings is the corroboration of positions that point to different ways of producing scientific knowledge.Es común en nuestra sociedad, incluso en situaciones escolares, hablarse de ciencia como si fuera única. Los resultados de la producción científica a menudo se valoran sin abordar la forma cómo se producen. De eso puede derivarse una posible imaginación sobre la existencia de un método científico único. Este artículo parte de la constatación de la relevancia de cuestionar posiciones que pueden valorar la ciencia y el método científico como si fueran únicos, es decir, posiciones que no se detienen en el carácter multidisciplinario de esa institución. Se busca comprender algunas representaciones de científicos y filósofos sobre ese tema, las cuales se infieren en la lectura de discursos presentes en recortes de sus producciones, siendo estos recortes seleccionados en vista de posibles reflexiones sobre el cuestionamiento señalado. Para el desarrollo de la lectura, así como para la noción de representación, nos apoyamos en los principios y nociones de análisis del discurso en la vertiente pecheutiano, principalmente en las producciones de Eni Orlandi. En esta vertiente, se considera el discurso el efecto de significados entre interlocutores. Entre los resultados de los análisis de discursos presentes en esos recortes está la corroboración de posiciones que apuntan a diferentes formas de producirse conocimientos científicos.É comum em nossa sociedade, inclusive em situações escolares, falar-se da ciência como se fosse única. Resultados da produção científica são, muitas vezes, valorizados sem que o modo como foram produzidos seja abordado. Um possível imaginário sobre a existência de um método científico único pode daí derivar. Este artigo parte da constatação da relevância de se questionarem posições que possam valorizar a ciência e o método científico como se fossem únicos, ou seja, posições que não se detêm no caráter multidisciplinar dessa instituição. Buscam-se compreender algumas representações de cientistas e filósofos sobre essa questão, as quais são inferidas na leitura de discursos presentes em recortes de suas produções, sendo esses recortes selecionados tendo-se em vista possíveis reflexões sobre o questionamento apontado. Para o desenvolvimento da leitura, bem como para a noção de representação, apoiamo-nos em princípios e noções da análise de discurso na vertente pecheutiana, principalmente em produções de Eni Orlandi. Nessa vertente, o discurso é considerado efeito de sentidos entre interlocutores. Entre os resultados das análises de discursos presentes nesses recortes destaca-se a corroboração de posições que apontam diferentes maneiras de se produzirem conhecimentos científicos

Experimental Chemotherapy for Chagas Disease: A Morphological, Biochemical, and Proteomic Overview of Potential Trypanosoma cruzi Targets of Amidines Derivatives and Naphthoquinones

Chagas disease (CD), caused by Trypanosoma cruzi, affects approximately eight million individuals in Latin America and is emerging in nonendemic areas due to the globalisation of immigration and nonvectorial transmission routes. Although CD represents an important public health problem, resulting in high morbidity and considerable mortality rates, few investments have been allocated towards developing novel anti-T. cruzi agents. The available therapy for CD is based on two nitro derivatives (benznidazole (Bz) and nifurtimox (Nf)) developed more than four decades ago. Both are far from ideal due to substantial secondary side effects, limited efficacy against different parasite isolates, long-term therapy, and their well-known poor activity in the late chronic phase. These drawbacks justify the urgent need to identify better drugs to treat chagasic patients. Although several classes of natural and synthetic compounds have been reported to act in vitro and in vivo on T. cruzi, since the introduction of Bz and Nf, only a few drugs, such as allopurinol and a few sterol inhibitors, have moved to clinical trials. This reflects, at least in part, the absence of well-established universal protocols to screen and compare drug activity. In addition, a large number of in vitro studies have been conducted using only epimastigotes and trypomastigotes instead of evaluating compounds' activities against intracellular amastigotes, which are the reproductive forms in the vertebrate host and are thus an important determinant in the selection and identification of effective compounds for further in vivo analysis. In addition, due to pharmacokinetics and absorption, distribution, metabolism, and excretion characteristics, several compounds that were promising in vitro have not been as effective as Nf or Bz in animal models of T. cruzi infection. In the last two decades, our team has collaborated with different medicinal chemistry groups to develop preclinical studies for CD and investigate the in vitro and in vivo efficacy, toxicity, selectivity, and parasite targets of different classes of natural and synthetic compounds. Some of these results will be briefly presented, focusing primarily on diamidines and related compounds and naphthoquinone derivatives that showed the most promising efficacy against T. cruzi

Estimativas mensais da formação bruta de capital fixo pública no Brasil (2002-2010)

This article presents new monthly estimates of the gross fixed capital formation of the Brazilian general government for the 2002-2010 years. A significant research effort was made to carry out the estimations. First, the many differences between the concepts of "public investment" (as used in the Brazilian public accounting manuals) and "gross fixed capital formation of the general government" (as used in the United Nations System of National Accounts) were carefully identified, and so were the peculiarities of the Brazilian budget process that distort official estimates of the annual gross fixed capital formation of the Brazilian general government and affect the precision of naive estimates of the same variable in higher frequencies. Second, several procedures were developed to correct or mitigate these problems. Finally, additional information was obtained from 20 out of 26 Brazilian states, the federal district, and tens of (large) municipalities in order to allow us to circumvent the relative scarcity of information on the finances of Brazilian state and local governments.Este artigo apresenta estimativas inéditas de séries mensais da formação bruta de capital fixo (FBCF) das administrações públicas no Brasil de 2002 a 2010. A estimação exigiu um esforço significativo de pesquisa. Em primeiro lugar, realizou-se uma análise criteriosa dos conceitos da contabilidade pública e da contabilidade nacional, enfatizando-se as peculiaridades do processo orçamentário brasileiro que enviesam as estimativas anuais dos investimentos públicos divulgadas nas estatísticas oficiais e que afetam a precisão das informações de alta frequência. Em segundo lugar, desenvolveram-se procedimentos para mitigar estes problemas. Por fim, buscou-se contornar a escassez de informações dos governos subnacionais solicitando-se informações adicionais de centenas de contadores públicos, técnicos ou secretários de Fazenda/Planejamento de todos os estados da Federação, do Distrito Federal (DF) e dos maiores municípios

Annona coriacea Mart. fractions promote cell cycle arrest and inhibit autophagic flux in human cervical cancer cell lines

Plant-based compounds are an option to explore and perhaps overcome the limitations of current antitumor treatments. Annona coriacea Mart. is a plant with a broad spectrum of biological activities, but its antitumor activity is still unclear. The purpose of our study was to determine the effects of A. coriacea fractions on a panel of cervical cancer cell lines and a normal keratinocyte cell line. The antitumor effect was investigated in vitro by viability assays, cell cycle, apoptosis, migration, and invasion assays. Intracellular signaling was assessed by Western blot, and major compounds were identified by mass spectrometry. All fractions exhibited a cytotoxic effect on cisplatin-resistant cell lines, SiHa and HeLa. C3 and C5 were significantly more cytotoxic and selective than cisplatin in SiHa and Hela cells. However, in CaSki, a cisplatin-sensitive cell line, the compounds did not demonstrate higher cytotoxicity when compared with cisplatin. Alkaloids and acetogenins were the main compounds identified in the fractions. These fractions also markedly decreased cell proliferation with p21 increase and cell cycle arrest in G2/M. These effects were accompanied by an increase of H2AX phosphorylation levels and DNA damage index. In addition, fractions C3 and C5 promoted p62 accumulation and decrease of LC3II, as well as acid vesicle levels, indicating the inhibition of autophagic flow. These findings suggest that A. coriacea fractions may become effective antineoplastic drugs and highlight the autophagy inhibition properties of these fractions in sensitizing cervical cancer cells to treatment.e FINEP (MCTI/FINEP/MS/SCTIE/DECIT-01/ 2013—FP XII-BIOPLAT), Barretos Cancer Hospital, CAPES, CNPq, FAPEMIG, UFSJ. RMR is a recipient of CNPq Productivity Gran

APLICAÇÃO DE CONTROLADORES FUZZY E PROPORCIONAL PARA UM ROBÔ SEGUIDOR DE PAREDE AUTÔNOMO EM AMBIENTE ESTÁTICO

A navegação de robôs móveis apresenta uma vari- edade de problemas, como a presença de ruídos e distúrbios, necessidade de fusão sensorial e a dificuldade na obtenção do modelo matemático que representa as interações físicas do ambiente. Cabe ao sistema de controle prover comandos para a correta execução dos movimentos do robô, de forma a alcançar algum objetivo proposto mesmo diante das adversidades do ambiente. Este artigo apresenta a aplicação de diferentes tipos de sistemas de controle, como controladores fuzzy convencional, fuzzy hierárquico e proporcional, para a navegação de um robô seguidor de parede em ambiente estático, cujo objetivo é evitar colisões e manter o robô a uma distância segura da parede. Foram feitas simulações em dois ambientes 3D e um experimento real para validação da abordagem proposta. As simulações foram feitas por meio do simulador V-REP em associação com a linguagem C++, utilizando um robô diferencial autônomo munido de sensores ultrassônicos. O experimento real fez uso de sensores e da placa Arduíno acoplados ao chassi de um robô diferencial. Os resultados mostram que o objetivo é alcançado de forma satisfatória por todas as técnicas propostas

Genome of the Avirulent Human-Infective Trypanosome—Trypanosoma rangeli

Background: Trypanosoma rangeli is a hemoflagellate protozoan parasite infecting humans and other wild and domestic mammals across Central and South America. It does not cause human disease, but it can be mistaken for the etiologic agent of Chagas disease, Trypanosoma cruzi. We have sequenced the T. rangeli genome to provide new tools for elucidating the distinct and intriguing biology of this species and the key pathways related to interaction with its arthropod and mammalian hosts.  Methodology/Principal Findings: The T. rangeli haploid genome is ,24 Mb in length, and is the smallest and least repetitive trypanosomatid genome sequenced thus far. This parasite genome has shorter subtelomeric sequences compared to those of T. cruzi and T. brucei; displays intraspecific karyotype variability and lacks minichromosomes. Of the predicted 7,613 protein coding sequences, functional annotations could be determined for 2,415, while 5,043 are hypothetical proteins, some with evidence of protein expression. 7,101 genes (93%) are shared with other trypanosomatids that infect humans. An ortholog of the dcl2 gene involved in the T. brucei RNAi pathway was found in T. rangeli, but the RNAi machinery is non-functional since the other genes in this pathway are pseudogenized. T. rangeli is highly susceptible to oxidative stress, a phenotype that may be explained by a smaller number of anti-oxidant defense enzymes and heatshock proteins.  Conclusions/Significance: Phylogenetic comparison of nuclear and mitochondrial genes indicates that T. rangeli and T. cruzi are equidistant from T. brucei. In addition to revealing new aspects of trypanosome co-evolution within the vertebrate and invertebrate hosts, comparative genomic analysis with pathogenic trypanosomatids provides valuable new information that can be further explored with the aim of developing better diagnostic tools and/or therapeutic targets

POTENCIAL ANTI-INFLAMATÓRIO DAS FOLHAS DE Chenopodium ambrosioides L. NO MODELO DE CISTITE HEMORRÁGICA EM CAMUNDONGOS

A cistite hemorrágica (CH) possui etiologia infecciosa, medicamentosa ou radioterápica. Consiste na presença de hematúria macroscópica secundária a sangramento vesical e uma das suas possíveis causas é o uso de ciclofosfamida (CYP). Várias alternativas farmacológicas têm sido investigadas para o tratamento da CH. Dentre as possibilidades, o potencial terapêutico de espécies vegetais tem sido avaliado. A espécie Chenopodium ambrosioides L. (Amaranthaceae), tem sido utilizado popularmente como anti-infamatório, efeito que  tem  sido comprovado cientifcamente. Assim, o objetivo deste estudo  foi investigar os efeitos do extrato bruto hidroalcoólico (EBH) de folhas secas de C. ambrosioides na CH induzida em camundongos pela ciclofosfamida. Camundongos fêmeas da linhagem Swiss receberam 150 mg/kg de CYP por via intraperitoneal para indução de CH. Em seguida, os animais foram tratados em dose única de acordo com protocolo estabelecido para cada grupo: soro fsiológico a 0,9% (grupo Controle); diclofenaco potássico (grupo Diclofenaco); EBH com dose única de 5 (grupo EBH5) ou 50mg/kg (grupo EBH50). Após 12 horas da indução da CH, o sangue dos animais foi retirado para realização do hemograma.  Os animais foram então sacrifcados e as bexigas retiradas, avaliadas macroscopicamente (hemorragia) e pesadas. Foram removidos, ainda, os órgãos linfóides a fm de realizar contagem de células do baço, medula óssea e linfonodos. Os resultados demonstraram que houve diminuição do peso das bexigas e da hemorragia nos grupos Diclofenaco e EBH5 quando comparados ao grupo controle. Houve um aumento das células da medula óssea, baço e linfonodo mesentérico em todos os animais tratados em relação ao controle. Em relação ao hemograma houve apenas aumentos pontuais no grupo EBH50. Em conclusão, o extrato bruto hidroalcoólico de folhas de C. ambrosioides na dose de 5mg/Kg apresentou efeito anti-infamatório e imunoestimulante, pois diminuiu o peso e a hemorragia da bexiga, e aumentou a produção e proliferação de células linfóides. Diante dos resultados desse estudo, bem como da evidência de ausência de toxicidade de outros trabalhos, podemos sugerir o tratamento com este extrato como alternativa terapêutica nos modelos de CH induzida por CYP em camundongos.Descritores: Anti-infamatório. Chenopodium ambrosioides. Ciclofosfamida. Cistite.AbstractHemorrhagic cystitis (HC) has infectious, drug or radiotherapy etiology. Consists in the presence of macroscopic hematuria secondary to bladder bleeding, and one of its possible causes is the use of cyclophosphamide (CYP). Several pharmacological alternatives have been investigated for the treatment of HC. Among the possibilities, the therapeutic potential of plant species have been reported. The species Chenopodium ambrosioides L. (Amaranthaceae) has been popularly used as an anti-infammatory efect that has been proven scientifcally. The objective of this study was to investigate the efects of crude hydroalcoholic extract of dried leaves of C. ambrosioides in HC cyclophosphamide induced in mice. Female mice of the Swiss strain received 150 mg / kg of CYP intraperitoneally to induce HC. Then the animals were treated with a single dose according to protocol established for each group: normal saline 0.9% (control group); diclofenac (diclofenac group); hydroalcoholic extract with a single dose of 5 (EBH5 group) or 50 mg / kg (EBH50 group). After 12 hours from the induction of HC, the bleeding was performed in the animal for the complete blood count. The animals were then sacrifced and had their bladders removed, as assessed macroscopically (bleeding) and weighed. The lymphoid organs were also removed in order to perform spleen, bone marrow and lymph nodes cell count. The results demonstrated that there was a decrease in the weight of bladders and bleeding in the diclofenac group and EBH5 when compared to the control group. There was an increase of cells in the bone marrow, spleen and lymph node in all treated animals as compared to control. In blood count there were only occasional increases in EBH50 group. In conclusion, the hydroalcoholic crude extract of Chenopodium ambrosioides leaves at a dose of 5 mg / kg showed anti-infammatory and immunostimulatory efect as decreased body weight and bleeding of the bladder, and increased production and proliferation of lymphoid cells. Given the results of this study, as well as evidence of absence of toxicity in other studies, we suggest treatment with this extract as an alternative therapy in models of CH-induced CYP in mice.Descriptors: Anti-infammatory. Chenopodium ambrosioides. Cyclophosphamide. Cystitis